The research program described in this application is focused on development of intracellular libraries of cyclic peptides as sources of molecular diversity for drug discovery and chemical genetics. Intracellular peptide and protein cyclization is made possible through circular permutation of inteins (internal proteins). Thus far we have used the technology to elaborate vast, chemically diverse libraries of low molecular weight cyclic peptide in bacterial host cells. To transform these libraries into a comprehensive, cross-cutting technology that can be translated into a variety of biomedical applications, we will: 1) demonstrate that intracellular cyclic peptide libraries can be used to generate high affinity ligands to a selected target receptor; 2) validate peptide cyclization technology in eukaryotic and mammalian cell hosts, which are most relevant for biomedical research; and 3) develop general strategies that enable facile identification of physiological targets that interact with cyclic peptides effectors in cells. My laboratory will pursue a protein engineering approach to these objectives, and implement the improvements as part of a program to discover small molecules that inhibit replication of hepatitis C virus (HCV). We will take advantage of existing libraries in bacterial hosts by creating a high throughput screen to identify potent inhibitors of a heterologously expressed HCV gene product (aim 1). We will optimize intein-mediated cyclization in mammalian host cells and evaluate compatibility of cyclization with signal sequence mediated subcellular localization (aim 2). We will modify the selection marker of the HCV replicon for greater versatility in chemical genetics studies of viral pathogenesis and transfer cyclic peptide inhibitors identified by screening bacterial libraries into the modified HCV replicon to evaluate their effects on viral replication in hepatocytes (aim 3). Finally, We will engineer cyclization constructs for compatibility with two hybrid systems, explore two hybrid systems, expression libraries and affinity- based methods for peptide target identification and initiate studies to identify new viral replication inhibitors and their physiological targets using intracellular libraries as tools for chemical genetics in the HCV replicon (aim 4). The integrated technology that will emerge from the work described in this proposal will provide a powerful new tool for the functional dissection of metabolic and signaling pathways in the physiological setting.